Currently, cartilage tissue engineering can yield tissue constructs with biomechanical properties comparable to native cartilage by using agarose, a hydrogel that is immunogenic and non-degradable. These properties seriously limit its studies in animal models and the use in humans. Even in vitro, the capability to modify and customize agarose structure, composition and mechanical properties is rather limited. Our laboratory has a long-standing interest and experience in using silk scaffolds, through collaboration with Dr. David Kaplan's laboratory. Recently, an innovative method was developed to generate silk hydrogels that are fully biocompatible and biodegradable. One particular preparation of silk hydrogel yielded cartilaginous constructs matching those based on agarose. As the structural and mechanical properties of silk hydrogel can be precisely controlled, we now have a tool, for the first time, to systematically study the factors and mechanisms underlying the suitability of hydrogels for cartilage tissue engineering. This discovery leads us to envision a functional cartilage tissue engineering system based on silk hydrogel. Preliminary results using bovine chondrocytes are extremely promising, with biomechanical properties of tissue constructs similar to those achieved using agarose hydrogel, and not achieved with any other scaffold. Taking advantage of the versatility of the silk material, we propose to examine the mechanisms behind the hydrogel's exceptional ability to promote cartilage tissue development. We will modify silk hydrogel properties via changes in silk extraction, concentration, and silk fiber reinforcement to vary material properties such as solute diffusivity and mechanical strength. By examining the resulting construct functionality, we expect to better understand the mechanisms behind cartilage matrix elaboration and assembly. Outcome of this proposal will also aid in optimization protocols in future material development. Our long-term goal is to generate an entire osteochondral construct using silk hydrogel and porous silk scaffolds to aid in cartilage tissue integration. PUBLIC HEALTH RELEVANCE: Cartilage tissue engineering is expected to result in human grafts suitable for implantation and thereby establish new treatment options for joint repair. The utility of such engineered grafts largely depends on their biomechanical properties, and most methods result in reasonably good biochemical compositions and limited load bearing ability of engineered grafts. One particular system, based on the cultivation of bovine chondrocytes in agarose hydrogel, has the ability to generate cartilage tissue constructs with properties similar to those of native cartilage. However, agarose is immunogenic and not degradable and therefore is not suitable for implantation. Also, the capability to modify and customize agarose structure, composition and mechanical properties is very limited. Our laboratory has a long standing interest in using silk as a biomaterial. One of the recently developed modifications of silk protein is a hydrogel which is, in contrast to agarose, biocompatible, degradable, and has properties in a wide range that can be tailored precisely. We discovered that one particular preparation of silk hydrogel can result in engineered cartilage with properties comparable to those achieved with agarose hydrogel. This discovery leads us to envision a functional cartilage tissue engineering system with silk hydrogel. Preliminary results using bovine chondrocytes are extremely promising, with constructs approaching functional properties similar to those found for agarose constructs. Taking advantage of the versatility of the silk material, we propose to examine the mechanisms behind the hydrogel's exceptional ability to promote cartilage tissue development. Outcome of this proposal will also aid in optimization protocols in future material development. The long-term goal of this research is also to generate an all-silk osteochondral construct using silk hydrogel and porous scaffold to aid in cartilage tissue integration.